Project Summary Despite remarkable achievements in the treatment, the rates of new HIV infections continue to grow, and AIDS remains the fifth largest morbidity in the world. Consequently, the development of an effective vaccine or combinatorial approaches that prevent HIV transmission is urgently needed. The RV144 vaccine trial, which used ALVAC-HIV vector and alum-adjuvanted AIDSVAX B/E gp120 protein, is the first, and so far the only, HIV vaccine to demonstrate limited but significant protection against HIV acquisition. While most HIV vaccines have been designed to elicit adaptive immunity, vaccine-mediated innate immune responses have been overlooked. HIV infection occurs mainly at the mucosal site, where localized foci are formed before the systemic spread of the virus. Thus, enhancing mucosal immunity would be an effective way to prevent infection. Increasing evidence shows the potential role of natural killer cells (NK) and innate lymphoid cells group-3 (ILC3) in HIV containment, and their ability to modulate adaptive immune responses and mucosal homeostasis. Mounting protective NK cell responses and enhancing mucosal immunity by augmenting the functions of ILC3 would be an effective way of preventing infection. However, little is known about HIV vaccine-induced peripheral NK cells and mucosal ILC3 responses. To our knowledge, no vaccine study has focused on modulating NK or ILC3 to improve protection from HIV infection. We have recapitulated the similar protection observed in the RV144 trial in rhesus macaques using the same vaccination strategy. My preliminary data demonstrate the induction of early and durable NK and ILC3 responses in peripheral and mucosal sites following vaccination and a correlation between IL-17 producing NKp44+ILCs in the gut with reduced risk of infection in the multivariate analysis. Thus, I hypothesize that the ALVAC-SIV gp120/alum vaccination, combined with augmented NK and ILC3 responses, would enhance vaccine efficacy against HIV/SIV infection. To test this hypothesis, I will investigate the effect of ALVAC-SIV gp120 prime boost vaccination strategy on peripheral and mucosal ILCs using C57BL/6 mice. Next, I will determine whether the ALVAC-HIV gp120/ alum prime-boost vaccination, combined with augmented mucosal immunity by modulating ILC3 function via interacting with Aryl hydrocarbon receptor (AhR) of ILC3 in the gut can enhance the protection against HIV infection using humanized BLT mice. The results of this study will enhance our understanding of the interplay between innate and adaptive responses in systemic and mucosal sites during HIV vaccine. I envision that the combination of a moderately protective vaccine with enhanced mucosal immunity via modulating NK/ILC3 by interacting with AhR will be recognized as a novel HIV preventive vaccine approach. These results will lead ultimately to the development of a protective HIV vaccine strategy for humans.